Smart-Seq

Switch Mechanism at the 5′ End of RNA Templates

Smart-Seq was developed as a single-cell sequencing protocol with improved read coverage across transcripts (Ramskold et al., 2012). Complete coverage across the genome allows the detection of alternative transcript isoforms and SNPs. There are 2 versions of Smart-Seq: Smart-Seq and Smart-seq2. Smart-seq2 includes several improvements over the original Smart-Seq protocol (Picelli et al., 2013)(Picelli et al., 2014). The new protocol includes a locked nucleic acid (LNA), an increased MgCl2 concentration, betaine, and elimination of the purification step to improve the yield significantly.

Smart-Seq: Cells are lysed, and the RNA is hybridized to an oligo(dT)-containing primer. The first strand of the cDNA is synthesized with the addition of a few untemplated C nucleotides. This poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer is hybridized to the poly(C) overhang and used to synthesize the second strand. Full-length cDNAs are PCR-amplified to obtain nanogram amounts of DNA. The PCR products are purified for sequencing.

Reviews:This method has been widely integrated into various sequencing techniques due to its high versatility.References:This method has been widely integrated into various sequencing techniques due to its high versatility.

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History: Smart-Seq

Smart-Seq was developed as a single-cell sequencing protocol with improved read coverage across transcripts (Ramskold et al., 2012). Complete coverage across the genome allows the detection of alternative transcript isoforms and SNPs. There are 2 versions of Smart-Seq: Smart-Seq and Smart-seq2. Smart-seq2 includes several improvements over the original Smart-Seq protocol (Picelli et al., 2013)(Picelli et al., 2014). The new protocol includes a locked nucleic acid (LNA), an increased MgCl2 concentration, betaine, and elimination of the purification step to improve the yield significantly.

Smart-Seq: Cells are lysed, and the RNA is hybridized to an oligo(dT)-containing primer. The first strand of the cDNA is synthesized with the addition of a few untemplated C nucleotides. This poly(C) overhang is added exclusively to full-length transcripts. An oligonucleotide primer is hybridized to the poly(C) overhang and used to synthesize the second strand. Full-length cDNAs are PCR-amplified to obtain nanogram amounts of DNA. The PCR products are purified for sequencing.

Reviews:This method has been widely integrated into various sequencing techniques due to its high versatility.References:This method has been widely integrated into various sequencing techniques due to its high versatility.

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